Figure 6.

Treatment of J774A.1 macrophages with ASX-loaded microparticles inhibits TGF$\beta$ secretion. (a) The cells were treated with ASX microparticles following the scheme sketched on top of the panel. At day 5 the supernatants from J774A.1 cells were administered to MBF-F11 cells and after 2 more days secreted alkaline phosphatase (SEAP) activity in the culture supernatants (SN) of these cells was measured. Bar labels refer to: ctrl −, untreated MBF-F11 cells; ctrl +, MBF-F11 cells treated with J774A.1 cells supernatants subjected to acid denaturation (to dissociate the TGF$\beta$-LAP complex and activate the cytokine); ctrl + no acid, in this case J774A.1 cells supernatants are not subjected to acid denaturation; P-ASX (t − 1, t0, t + 1, t + 2), treatment of J774A.1 cells with ASX microparticles at the time points indicated on top of the panel. The differences in the data shown in this panel are statistically significant ($F_{5,11}=41.8$, $p=8.6\times {10}^{-7}$, ANOVA test). Only ASX particles given at day 3 and 4 after J774A.1 cell seeding significantly inhibit TGF$\beta$ secretion ($p=0.027$ and $p=0.02$, respectively, Tukey-HSD post-hoc test). (b) The cells were treated with ASX microparticles following a slightly modified scheme with respect to that described above. As shown in the diagram on top of the panel, at day 2 the cells were also treated with two different concentrations of IFN$\gamma$ (interferon gamma). Inhibition of TGF$\beta$ secretion by ASX microparticles is greater than that observed in panel (a), an effect that is probably correlated to the higher uptake of the particles in IFN$\gamma$-treated macrophages (see, e.g., Figure 2). Bar labels are as in panel (a) with the following additions: IFN no acid, MBF-F11 cells treated with supernatants, not subjected to acid denaturation, from IFN$\gamma$-stimulated J774A.1 cells; IFN, MBF-F11 cells treated with acid-denatured supernatants from IFN$\gamma$-stimulated J774A.1; Pt0+IFN, MBF-F11 cells treated with acid-denatured supernatants from J774A.1 cells treated with IFN$\gamma$ and empty microparticles. Treatment with IFN$\gamma$ at 50 ng/mL does not significantly increase bioactive TGF$\beta$ secretion by J774A.1 cells but significantly increases the inhibitory effect of ASX microparticles administered at day 1 and 2 after J774A.1 cell seeding ($p=0.006$ and $p=0.001$, respectively, Tukey-HSD post-hoc test). Treatment with IFN$\gamma$ at 100 ng/mL do not significantly increase bioactive TGF$\beta$ secretion by J774A.1 cells but significantly increases the inhibitory effect of ASX microparticles administered at day 0, 1, and 2 after J774A.1 cell seeding ($p=0.04$, $p=0.003$ and $p=0.0006$, respectively, Tukey-HSD post-hoc test).